RESEARCH ARTICLE 1863

Development 139, 1863-1873 (2012) doi:10.1242/dev.074005 © 2012. Published by The Company of Biologists Ltd Identification of molecular compartments and genetic circuitry in the developing mammalian Jing Yu1,2, M. Todd Valerius1,*, Mary Duah1, Karl Staser1, Jennifer K. Hansard1, Jin-jin Guo1, Jill McMahon1, Joe Vaughan1, Diane Faria1, Kylie Georgas3, Bree Rumballe3, Qun Ren2, A. Michaela Krautzberger1, Jan P. Junker4, Rathi D. Thiagarajan3, Philip Machanick3,‡, Paul A. Gray5,§, Alexander van Oudenaarden4, David H. Rowitch6, Charles D. Stiles7, Qiufu Ma5, Sean M. Grimmond3, Timothy L. Bailey3, Melissa H. Little3 and Andrew P. McMahon1,¶

SUMMARY Lengthy developmental programs generate cell diversity within an organotypic framework, enabling the later physiological actions of each organ system. Cell identity, cell diversity and cell function are determined by cell type-specific transcriptional programs; consequently, transcriptional regulatory factors are useful markers of emerging cellular complexity, and their expression patterns provide insights into the regulatory mechanisms at play. We performed a comprehensive genome-scale in situ expression screen of 921 transcriptional regulators in the developing mammalian urogenital system. Focusing on the kidney, analysis of regional-specific expression patterns identified novel markers and cell types associated with development and patterning of the . Furthermore, promoter analysis of synexpressed predicts transcriptional control mechanisms that regulate cell differentiation. The annotated informational resource (www.gudmap.org) will facilitate functional analysis of the mammalian kidney and provides useful information for the generation of novel genetic tools to manipulate emerging cell populations.

KEY WORDS: Genome-scale expression screen, Transcriptional regulator, Kidney, Mouse

INTRODUCTION that cap each branch tip (Carroll et al., 2005; Kobayashi et al., The mammalian metanephric kidney is essential for the 2008; Park et al., 2007). At each round of branching, a subset of maintenance of water and electrolyte homeostasis, the regulation these progenitors undergoes an epithelial transition that establishes of blood pressure and blood cell composition, and bone formation a renal vesicle (RV). RVs undergo elaborate patterning and (Koeppen and Stanton, 2001). Development of a functional kidney morphogenesis along a glomerular-collecting duct axis that is requires a complex interplay among its principal cellular crucial for organ function. A highly developed vascular network components (Costantini and Kopan, 2010; Dressler, 2009). (Gomez et al., 1997; Woolf and Loughna, 1998), interstitial support The nephric duct-derived ureteric forms the arborized cells and a variety of distinct subregions with specialized properties network of the in response to branching that include contractile and sensory functions all contribute to the signals derived from adjacent . This network is crucial physiological actions of the kidney. for urine transport from the to the , and the Not surprisingly, given the central role of the kidney in human maintenance of pH and osmolarity in tissue fluids. Signals from the health, the experimental analysis of mammalian kidney nascent ureteric epithelium regulate survival, proliferation and development has received considerable attention. The kidney was differentiation of mesenchymal, stem cell-like, nephron progenitors the first mammalian organ shown to replicate a developmental program in culture (Grobstein, 1953; Grobstein, 1955; Saxen, 1987). Since those pioneering studies in the 1950s, a wealth of 1Department of Stem Cell and Regenerative Biology, Department of Molecular and genetic, molecular and cellular information has informed on the Cellular Biology, Harvard Stem Cell Institute, Harvard University, 16 Divinity Avenue, control of kidney development (Costantini and Kopan, 2010; Cambridge, MA 02138, USA. 2Department of Cell Biology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA. 3Institute for Molecular Dressler, 2009). Collectively, these studies have underscored the Bioscience, University of Queensland, St Lucia 4072, Australia. 4Department of complexity of kidney development, and highlighted the need for a Physics and Biology, Massachusetts Institute of Technology, 77 Massachusetts more systematic analysis of cell types, cell relationships and cell 5 Avenue, Cambridge, MA 02139, USA. Department of Neurobiology, Harvard interactions in the assembly of the kidney. The increasing evidence Medical School, Boston, MA 02115, USA. 6Departments of Pediatrics and Neurological Surgery, Howard Hughes Medical Institute, University of California San that developmental deficiencies increase the risk of kidney disease Francisco, 35 Medical Center Way, San Francisco, CA 94143, USA. 7Department of in later life emphasizes the need to enhance our understanding of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115, developmental events (Bertram et al., 2011; Song and Yosypiv, USA. 2011). *Present address: Department of Surgery, Transplant Institute, Beth Israel Deaconess One barrier to progress is a dearth of molecular markers that can Medical Center, Harvard Medical School, Boston, MA 02115 USA distinguish cell types and facilitate analysis of developmental ‡Present address: Department of Computer Science, Rhodes University, Grahamstown, 6140, South Africa programs. In many systems, transcriptional regulators have served §Present address: Department of Anatomy and Neurobiology, Washington University as important cell type-specific markers and drivers of cell School of Medicine, 660 S. Euclid Avenue, St Louis, MO 63110-1093, USA specification and differentiation. For example, large-scale ¶Author for correspondence ([email protected]) expression screens of transcriptional regulators have proven fruitful

Accepted 1 March 2012 in identifying organizational and developmental features in the DEVELOPMENT 1864 RESEARCH ARTICLE Development 139 (10) assembly of the mammalian nervous system (Gray et al., 2004). In situ hybridization of whole-mount urogenital systems with We surmised that systematic identification of the expression digoxigenin-labeled riboprobes patterns of a large set of transcriptional regulators in the developing For whole-mount in situ hybridization, E15.5 urogenital system samples urogenital system would provide new insights into the emergence were rehydrated through a graded series of methanol/NaCl. After of cell heterogeneity central to kidney formation and function. proteinase K (10 mg/ml) treatment for 30 minutes, samples were fixed Furthermore, the accrued data could potentially facilitate in silico again and prehybridized at 70°C for 1 hour before being hybridized with 500 ng/ml digoxigenin-labeled riboprobes at 70°C overnight. All steps prediction of networks, and enable the design of novel genetic post-hybridization were performed with a BioLane HTI liquid handling approaches for analysis of key cell types. system (Intavis Bioanalytical Instruments AG, Widdersdorferstrasse, Here, we report on a genome-scale in situ analysis of the Koeln, Germany). Briefly, samples were washed with hot solutions at 65°C expression of genes encoding mammalian transcriptional and treated with 100 mg/ml RNase A for 1 hour. After additional stringent regulatory factors in the developing mouse urogenital system. Our hot washes, samples were washed with 1ϫMBST [0.1 M maleic acid, 0.15 analysis provides a wealth of new markers of kidney development M NaCl, 0.1% Tween-20 (pH 7.5)], blocked in blocking solution (10% and reveals novel molecular subdomains in developing renal sheep serum in MBST plus 2% Boehringer Mannheim Blocking Reagent) structures. A meta-analysis of selected transcriptional regulators for 3-4 hours at room temperature, and incubated subsequently with anti- highlights the potential of this dataset for discovery of networks digoxigenin antibody conjugated with alkaline phosphatase (AP) (Roche, governing differentiation of terminal cell fates. Indianapolis, IN, USA, 1:4000) overnight at 4°C. Extensive washing in 1ϫMBST was followed by incubation in NTMT [100 mM NaCl, 100 mM MATERIALS AND METHODS Tris-HCl (pH 9.5), 50 mM MgCl2, 0.1% Tween-20, 2 mM levamisole). Further details of the protocols described below can be found Samples were then removed from the BioLane HTI, and the hybridization at http://www.gudmap.org/Research/Protocols/McMahon.html and signal visualized by adding BM purple. Color reactions were terminated at http://www.gudmap.org/Research/Protocols/Little.html. All experimental 3-hour, 6-hour, 9-hour, 12-hour, 24-hour, 36-hour and 48-hour time points, procedures with mice were performed in accordance with institutional and when signals were strong or a background developed. After development national animal welfare laws, guidelines and policies, and were approved of the colorimetric assay, samples were post-fixed, cleared through a by relevant institutional animal care committees. graded series of glycerol and stored in 80% glycerol/PBS. Images were then captured with a Nikon DXM1200 digital camera (Nikon, Melville, Plasmid preparation and riboprobe production NY, USA), attached to a Nikon SMZ1500 stereoscope (Nikon, Melville, Plasmid glycerol stocks from the Brain Molecular Anatomy Project NY, USA) at two different magnifications to view the entire urogenital (BMAP) cDNA library or selected cDNAs from Open Biosystems system and for a higher resolution view of the kidney. (Huntsville, AL, USA) were streaked on LB (Amp) plates and grown at 37°C overnight. Three colonies per clone were restreaked onto ‘master In situ hybridization and double in situ hybridization on frozen plates’. Plasmid DNA was purified from one colony per clone and sections with digoxigenin-labeled and fluorescein-labeled sequenced to confirm the expected sequence, and determine the orientation riboprobes of the cDNA. cDNA inserts were amplified from their plasmid backbone For frozen section in situ hybridization, kidney sections were post-fixed in with polymerase chain reaction (PCR) using T7, T3 or SP6 primers 4% PFA. After treatment with 10 mg/ml proteinase K for 10 minutes, depending on the vector backbone. sections were fixed again with 4% PFA, acetylated and dehydrated. A large number of transcriptional regulator-specific probes were Sections were then incubated with 500 ng/ml digoxigenin-labeled generated from a previously described collection (Gray et al., 2004). For riboprobes at 68°C overnight. For double in situ hybridization, sections these, cDNA inserts were amplified by PCR from a 1:100 dilution of were incubated with both digoxigenin-labeled and fluorescein-labeled Qiagen minipreps of cDNA plasmid subclones as above. An additional riboprobes. Sections were washed after hybridization and treated with 2 source of cDNA templates were provided by Fantom3 cDNA clones mg/ml RNase A for 15 minutes at 37°C. After stringent hot washes, punched from a RIKEN DNAbook 2 (Source BioScience imaGenes, sections were blocked for 1 hour or longer and then incubated with anti- Berlin, Germany). PCR primers were designed to amplify a 500-700 bp digoxigenin-AP overnight at 4°C. After washing in 1ϫMBST and region of the open reading frame for a given clone; a T7 promoter sequence equilibration in NTMT, sections were incubated with BM purple and the (TAA TAC GAC TCA CTA TAG GG) appended to the 3Ј clone-specific slides developed for a maximum of 7 days. Following color reaction, primer enabled direct generation of antisense riboprobes from T7-mediated sections were fixed and slides mounted with Glycergel mounting medium transcription of PCR products. Finally, for manual cloning of cDNA inserts (DAKO, Carpinteria, CA, USA). Images were captured with a Leica for probe production, specific primers were designed in Vector NTI MZ16F stereoscope equipped with a DFC300 FX camera (Leica, Buffalo (Invitrogen, Carlsbad, CA, USA) that would amplify ~750 bp of Refseq Grove, IL, USA). For double in situ hybridization, after the BM purple annotated cDNA sequence encoding the target regulatory factor of interest, color reaction, sections were re-fixed with 4% PFA for 30 minutes, washed using the T7 3Ј primer strategy above for direct synthesis of antisense with 1ϫMBST and incubated overnight at 4°C with an anti-Fluorescein- riboprobes. Riboprobes were purified with Microspin columns (BioRad, AP conjugated antibody that had been preabsorbed with mouse embryo Hercules, CA, USA) and diluted with prehybridization buffer [50% powder to eliminate non-specific activity. After washing in 1ϫMBST and formamide, 5ϫSSC (pH 4.5), 50 mg/ml yeast tRNA, 1% SDS, 50 mg/ml equilibration in NTMT, sections were incubated with INT/BCIP+10% Heparin] to 10 mg/ml. polyvinyl alcohol (PVA) to visualize hybridization of the second probe over a development period that extended for a maximum of 7 days. The Preparation of urogenital system samples slides were then fixed and mounted in Glycergel mounting medium For whole-mount in situ hybridization, the entire mouse urogenital system (DAKO, Carpinteria, CA, USA). with associated adrenal glands was dissected free of other embryonic tissues at embryonic day 15.5 (E15.5, TS23) and fixed in 4% In situ hybridization on paraffin sections paraformaldehyde (PFA) in PBS for 24 hours at 4°C. The specimens were The paraffin section in situ hybridization protocol has been previously dehydrated through a graded series of methanol/NaCl and stored in 100% published (Georgas et al., 2008; Little et al., 2007; Rumballe et al., 2008). methanol at –20°C before in situ hybridization. For each gene examined by section in situ hybridization, a DNA template For section in situ hybridization, E15.5 kidneys and were of 500-1000 bp was generated from either cDNA or plasmid clones by dissected free of all surrounding tissues, fixed in 4% PFA for 24 hours at PCR and transcribed to produce a digoxigenin (DIG)-labeled antisense 4°C, and cryopreserved in 30% sucrose overnight. Samples were riboprobe (www.gudmap.org). The primer and probe sequences are embedded in OCT, flash frozen in an ethanol/dry-ice bath and stored at available on the GUDMAP website within the section in situ hybridization

–80°C. Blocks were sectioned at 20 mm for section in situ hybridization. data submissions for each gene. Kidneys were harvested from E15.5 CD1 DEVELOPMENT Transcription factors in kidney RESEARCH ARTICLE 1865 mice and fixed overnight in 4% PFA at 4°C (Ethics analyzed in Genespring version 7.3 (Agilent, Santa Clara, CA, USA). One- IMB/180/10/NHMRC/NIF NF). Kidneys were paraffin embedded and way-ANOVA (P<0.01) with FDR correction (Benjamini-Hochberg) was sectioned at 7 mm. Slides were dewaxed manually and the remaining performed to identify differentially expressed probe sets across the entire section in situ hybridization performed using a Tecan Freedom Evo 150 dataset that was then used for subsequent analyses. Probe sets representing platform. Sections were hybridized at 64°C for 10 hours with 0.5-1.0 mg/ml candidate target genes were required to have synexpression to the of riboprobe, washed and incubated with anti-DIG-alkaline phosphatase probe set of interest, based on a minimum Pearson Fab fragments (1:1000, Roche, Indianapolis, IN, USA) for 120 minutes at correlation similarity measure of 0.7. Where a transcription factor probe 25°C. Detection of alkaline phosphatase activity using BM Purple (Roche, set in microarray did not correlate with in situ hybridization annotated Indianapolis, IN, USA) was performed manually. The signal intensity was expression (Pou3f1), we used a directed analysis approach specifying the monitored for up to 120 hours and the slides rinsed and fixed in 4% PFA required compartment-specific expression for target genes (FC≥2). prior to mounting in aqueous mounting medium. Images were captured with either an Olympus dotSlide System (Olympus, Mt Waverley, VIC, Prediction of transcription factor targets based on promoter Australia) or a standard light microscope (Olympus BX51 DP70 color 12 analysis megapixel digital camera, Mt Waverley, VIC, Australia). For each transcription factor analyzed, a defined binding motif was identified from one of the Uniprobe (Newburger and Bulyk, 2009), Jaspar (Portales- Annotation of Casamar et al., 2010) and TRANSFAC (Matys et al., 2006) motif databases. Expression patterns observed in all whole-mount in situ hybridization and The promoters of potential targets were defined as extending from –1500 bp section in situ hybridization samples were annotated against a previously to +500 bp relative to the genomic location of each given target site. To described comprehensive ontology of the developing urogenital tract (Little predict statistically the most likely potential targets regulated by the selected et al., 2007). Images and annotations are housed at www.gudmap.org. transcription factors, binding sites (TFBS) were predicted using Monkey (Moses et al., 2004), which uses species conservation to predict binding sites Single molecule fluorescence in situ hybridization (FISH) for a given motif. We have found Monkey to be a reliable TFBS predictor For each mRNA examined, a set of 48, 20-mer DNA oligonucleotides (Hawkins et al., 2009). Monkey calculates the probability that a given motif Ј complementary to coding and 3 untranslated regions were designed using binds to specific locations in a given set of promoters, using conservation an online available program (http://www.singlemoleculefish.com/ and comparing the match of the motif to each site in the promoter sequences Ј designer.html) and synthesized with 3 -amino modifications by Biosearch against that of randomly shuffled motifs. Monkey was preformed within a Technologies. Probe sets were pooled at a concentration of 1 nM for each previously described pipeline specialized to this kind of prediction (Piper et oligonucleotide, dried (Speedvac, medium heat), resuspended in 0.1 M al., 2010). As well as using the transcription factor motif and promoter sodium bicarbonate (pH 8.5) containing an excess of succinimidyl ester sequences, Monkey uses a sequence alignment of the promoters with derivatives of Alexa594 (Invitrogen, Carlsbad, CA, USA) or Cy5 (GE orthologous species. In this study, we generated data for two distinct sets of Healthcare, Piscataway, NJ, USA), and incubated overnight at room ortholog comparisons. In the first, mouse (mm9) genome was aligned with temperature with gentle agitation. Excess fluorophore was removed by rat (rn4), guinea pig (cavPor2) and rabbit (oryCun1), a phylogenetic tree ethanol precipitation, and coupled oligonucleotides were separated from containing the orthologous species, with a 0-order background Markov the uncoupled fraction by reverse phase high-pressure liquid model for mouse sequence data, with a motif database to ensure that shuffled chromatography. motifs generated to test significance of the motif of interest are not real motifs Kidneys obtained from intercrosses of Hoxb7Cre transgenic mice (Yu et and with a number of shuffled motifs to compare with the predicted motif. al., 2002) to ROSA-mT/mG double fluorescent reporter allele A second comparison was performed between mouse (mm9), rat (rn4), tm4(ACTB-tdTomato-EGFP)Luo [Gt(ROSA)26Sor /J] (Muzumdar et al., 2007) were human (hg18) and zebrafish (danRer5). Both resulted in a statistical dissected at E15.5, fixed in 4% PFA for 1 hour at 4°C, and incubated prediction for each promoter analyzed, based upon the most conserved overnight in 30% sucrose at 4°C before embedding in OCT compound. predicted TFBS between all orthologs (see supplementary material Table S3). Tissue blocks were sectioned at 6 mm, fixed in 4% PFA at room temperature for 15 minutes, rinsed in PBS and incubated overnight in 70% ethanol at 4°C. RESULTS The detailed hybridization procedure is documented elsewhere (Raj et In situ hybridization of known genes identified al., 2008). Briefly, sections were rehydrated in wash buffer before eight readily discernible expression patterns hybridizing with ~0.3 ng/ml of labeled probe sets for each mRNA overnight at 30°C in the dark. Sections were washed twice for 30 minutes; DAPI correlating with distinct cellular compartments in (Invitrogen, Carlsbad, CA, USA) was added in the second wash to enable the E15.5 kidney and ureter nuclear visualization and sections were mounted in anti-bleach buffer. Z- By E15.5, the ureteric network has branched eight or nine times stack images were taken at 0.3 mm intervals with a Nikon Ti Eclipse (Cebrian et al., 2004) and an elaborate ductal tree is established. inverted fluorescence microscope (Nikon, Melville, NY, USA) equipped Development of the is under way, and the renal with a 100ϫ oil-immersion objective and a Roper Scientific Pixis CCD medullary zone, which is crucial for concentrating urine, is starting camera using MetaMorph software (Molecular Devices, Sunnyvale, CA, to emerge (Cebrian et al., 2004; Yu et al., 2009). Nephrogenesis is USA). For semi-automated counting of particles, images were filtered and advancing at this time: in addition to early stage renal vesicles and processed as previously described (Raj et al., 2008). Spearman correlation comma- and S-shaped bodies, late stage renal corpuscles, proximal coefficients of pairs of genes were calculated in Matlab. To obtain P- values, these correlation coefficients were compared with the correlation and distal convoluted tubules, and early stage loops of Henle coefficients of 104 randomized datasets in which the values for one of the (LOHs) are present, which is indicative of maturing . genes were shuffled among cells. The Z-scores of the experimental Consequently, analysis at E15.5 enables the identification of early correlations compared with those of the randomized datasets were and late emerging cell types at a time-point technically suited for a transformed to P-values based on the normal distribution. sensitive three-dimensional, low-resolution perspective of in situ gene expression through whole-mount in situ hybridization. The Selection of potential transcription factor targets based upon high-throughput whole-mount in situ hybridization primary screen synexpression across the developing kidney expression atlas Target genes with synexpression to the compartment-specific transcription provides a broad overview of the expression of transcriptional factor of interest were identified through an expression profile similarity regulatory components throughout the entire urogenital system. measure using the GUDMAP embryonic kidney subcompartment We first optimized whole-mount in situ hybridization around a microarray atlas (Brunskill et al., 2008) (www.gudmap.org) (GEO: series of genes that unambiguously score most major renal

GSE6290). The microarray expression data was RMA normalized and components at E15.5: Eya1, Wnt4, Slc12a1, Wnt11, Wnt7b, Foxd1, DEVELOPMENT 1866 RESEARCH ARTICLE Development 139 (10)

categories: cap mesenchyme, early tubules (including pretubular aggregate, renal vesicle, comma- and S-shaped bodies), late tubules (nephrons beyond the S-shaped body stage), ureteric tip, ureteric trunk, renal interstitium, renal vasculature and ureter (including the ureter and the renal pelvis). These expression patterns, schematized in Fig. 1, form the basis of our subsequent comprehensive annotation of the expression of transcriptional regulatory factors.

A comprehensive screen of the expression of genes encoding transcriptional regulatory factors identified molecularly distinct domains in the embryonic kidney and ureter From analysis of two independently generated genome-wide compilations of mammalian transcriptional regulators (Gray et al., 2004; Lee et al., 2007), we compiled a target list of 951 transcriptional regulatory factors for which there was a general consensus of this classification (see supplementary material Table S1). We performed whole-mount in situ hybridization on 921 of these genes (96.8% coverage) and annotated expression in regard to the annotation groupings documented in Fig. 1. Our emphasis has been to provide an accurate account of expression patterns that can be unambiguously discerned. The whole-mount in situ hybridization approach is subject to false-negative results; for example, where weak internal signals are masked by stronger superficial ones. And whole-mount in situ hybridization does not enable a high-resolution description of all expression domains – an outcome that can only be realized through serial-section, multi- probe based section in situ hybridization analysis. What the whole- mount in situ hybridization does generate is a broad framework of the expression of potential regulatory factors that greatly facilitates secondary analyses. All whole-mount in situ hybridization images and their annotations can be searched, viewed and mined at www.gudmap.org. Of the large set of factors, 213 (23.1%) displayed regionally restricted expression in the kidney (bold genes in supplementary material Table S1) whereas 337 (36.6%) appeared to be ubiquitously expressed. In particular, we identified 106 (11.5%) genes expressed in only one renal category (excluding the ureter). Representative expression patterns of genes with localized expression are presented in Fig. 2, along with information on the Fig. 1. Eight characteristic and readily discernible gene number of genes displaying a given pattern, and the subset of these expression patterns are identified in the E15.5 kidney and ureter genes whose expression is unique to the domain of interest. Section by in situ hybridization and associated with specific annotation terms (groups). Whole-mount in situ hybridization analysis (left in situ hybridization was performed on a subset of genes in each panels) of E15.5 whole-mount kidneys and ureters reveals anatomically category to validate expression with cellular resolution, and to distinct expression profiles for eight kidney genes that are further reveal expression domains potentially masked by whole-mount in resolved through high-resolution section in situ hybridization analysis situ hybridization (Figs 2, 3). (center panels). Together, these eight probes mark many of the major cell types/structures of the kidney and ureter (schematized in the right Examination of complex transcription factor panels). When a gene is expressed in both superficial and internal expression patterns identifies novel domains of structures, signals from superficial structures may mask internal signals regulatory activity and cellular subcompartments in whole-mount in situ hybridization analysis. Arrows indicate The majority of documented expression patterns do not correspond expression associated with the respective annotation terms. Scale bar: to a specific anatomically defined compartment. For example, 200mm. section in situ hybridization on a subset of 65 genes with whole- mount in situ hybridization expression patterns annotated to early Sox17 and Shh (Fig. 1). As anticipated, each probe generated a tubules (supplementary material Table S1) revealed domains within distinctive and readily identifiable expression pattern, validated the S-shaped body that do not readily match proximal, medial and through high-resolution section in situ hybridization analysis (Fig. distal segments defined in classical histological studies (Little et al., 1). Deep structures such as the ureteric tree (Wnt7b, Shh) and thick 2007). Rather, the data point to a finer patterning of the S-shaped ascending limb of the LOH (Slc12a1) were uniformly labeled, and body, with molecular subdivisions of each segment (Fig. 3). Both the technique was sufficiently sensitive to detect weak expression Osr2 and Irx1 are expressed in the medial segment of the S-shaped of Shh in the ureteric tree, deep within the kidney. The observed body; however, their expression domains do not span the entire

expression patterns could be readily grouped into eight broad medial segment, and may not be precisely congruent (Fig. 3A). DEVELOPMENT Transcription factors in kidney RESEARCH ARTICLE 1867

Fig. 2. Expression of genes encoding transcriptional regulators within each annotation group. Low- and high- magnification whole-mount in situ hybridization images (left panels) display transcriptional regulators whose expression is restricted to one of the eight characteristic expression domains: the number of transcriptional regulators showing unique expression in each of these domains versus the total number of genes with a specific annotation to each term is shown (far right). Complementary low- and high-magnification section in situ hybridization images for each gene of interest provide cellular resolution of expression domains (right panels). ‘Unique’ genes may also be expressed in extrarenal compartments within the urogenital system. Most genes showing regionally restricted expression are expressed in more than one annotation group. Arrows indicate expression in ureteric tips. Scale bars: 200mm (low magnification); 20mm (high magnification).

Similarly, although both Tcfap2b and Pou3f3 (Brn1) are expressed distinct cellular boundaries within the ureteric epithelium. Sox8 in distal and medial segments of the S-shaped body, Pou3f3 transcripts localized to each tip in a bifurcating branch (Fig. 3C), expression extends slightly proximally to Tcfap2b (Fig. 3A). Pou3f3 whereas Emx2 expression within the branches was almost is reportedly expressed in both limbs of the loop of Henle (LOH) complementary to Sox8 expression (Fig. 3C), providing evidence anlage (Nakai et al., 2003), whereas data here suggest that Tcfap2b for the action of distinct transcriptional programs within the context is expressed only in the ascending limb of the extending LOH (Fig. of a highly dynamic branching process that may, for example, 3B). This raises the possibility that the proximal boundary of Tcfap2b segregate cells between tip stem/progenitor and stalk domains. marks the boundary between the ascending and descending limbs of Conversely, 19 transcriptional regulators were expressed the LOH, suggesting that the two limbs of the LOH may be specifically in the ureteric trunk but not the tip (supplementary differentially specified from the onset of LOH formation. Genetic material Table S1). Of these, only one gene, Foxi1, exhibited a fate mapping of these expression domains relative to the functional variegated expression pattern in a subset of ureteric trunk cells, anatomy of the adult kidney will likely provide complementary more readily discernible on section in situ hybridization analysis insights into how nephron complexity is generated. (Fig. 3D), concurring with a previous report of Foxi1 action in the Within the ureteric epithelium, 25 transcriptional regulators were mosaic differentiation of intercalated cells within the collecting identified with a whole-mount in situ hybridization expression duct epithelium (Blomqvist et al., 2004). pattern restricted to the ureteric tips and not detected in ureteric A large group of transcriptional regulators were annotated to the trunks, regardless of their expression in other components of the ureter and the renal pelvis (Fig. 2), most probably an overestimate kidney (supplementary material Table S1). Although all of these that reflects background from probe and/or antibody trapping within genes were excluded from the ureteric trunks, their whole-mount these structures. However, a close examination of a select group of

in situ hybridization expression patterns point to molecularly genes revealed previously unappreciated subdivisions in the DEVELOPMENT 1868 RESEARCH ARTICLE Development 139 (10)

Fig. 3. A selection of expression patterns potentially marking new compartment boundaries within the developing nephron, the ureteric epithelium and ureter. Primary whole-mount in situ hybridization analysis is shown in the left panels and high- and low-magnification section in situ hybridization in right panels. (A)Molecular subdivision of the S-shaped body from genes showing an early tubule annotation. Within the medial segment of the S-shaped body (SSB), Osr2 and Irx1 appear to display non-overlapping expression domains. The proximal boundary of Tcfap2b expression in the distal segment of the SSB (line) appears to lie slightly distal to that of Pou3f3 (line). The S-shaped bodies with their attaching ureteric epithelium are outlined in the high-magnification images and the expression domains of the genes are illustrated in the schematic drawings (far right). (B)Comparative expression of Tcfap2b and Pou3f3 in the loop of Henle (LOH) anlage suggests that Tcfap2b expression is restricted to a single arm of the LOH: the distal ascending limb (red arrows). (C)Differential expression of Sox8 and Emx2 in the bifurcating ureteric epithelium. (D)Ureteric trunk patterning and differentiation, as illustrated by the divergent expression domains of Foxi1 (intercalated cells of the collecting duct epithelium) and Foxa1 (urothelial lining of the pelvis and medullary collecting duct). (E)Prrx1 labels outer layers of loose ureteral mesenchyme. Scale bars: 200mm.

pelvic/medullary region and the ureter. For example, whole-mount epithelium (Thiagarajan et al., 2011). Whether the Foxa1 component in situ hybridization expression of Foxa1 in the urothelium and renal prefigures a segmental patterning of the medullary collecting ducts pelvic epithelium was confirmed by section in situ hybridization or plays an active role in medullary cell specification remains to be analysis (Fig. 3D), in addition, section in situ hybridization determined, but collectively the data suggest an early molecular examination showed an expression of Foxa1 in prospective stratification of epithelial structures prefiguring the cortico-medullary medullary collecting ducts, the segment of the collecting ducts axis of kidney organization and function. closest to the renal pelvic space (Fig. 3D). An independent study of The ureter consists of the urothelium and the ureteral microarray profiling results coupled with section in situ hybridization mesenchyme, including the lamina propria, the ureteral smooth

validation reported a similar molecular stratification of the ureteric muscle and the adventitia. At E15.5, the ureteral mesenchyme DEVELOPMENT Transcription factors in kidney RESEARCH ARTICLE 1869

Fig. 4. Vasculature-associated gene expression and expression of genes with broad sporadic expression in the developing kidney. (A)Selected genes with vasculature-associated expression patterns. (a-f)Bcl6b is weakly expressed in the cortex and medulla. (c-f)High magnification images showing its expression in individual cells in the renal cortex (red arrow) and cells invading the lower cleft of the S-shaped body (red arrow) (c), the glomerular capillary of the capillary loop stage renal corpuscle (d, red arrow), and the peritubular capillaries (e, red arrow). Bcl6b is also expressed in the endothelial cells of the arterioles and renal arteries (red arrows) but not that of the renal veins (yellow arrow) (f). Panels c and f are from a different tissue section from that of panel b. (g-l)Heyl is expressed in the renal arterial (h, red arrow) and arteriolar (i, red arrows) smooth muscles, but not the arterial and arteriolar endothelium (h,i, yellow arrows). It is also expressed in the mesangium of the capillary loop stage renal corpuscle (j, red arrows) but decreased to almost undetectable levels in that of the immature renal corpuscle (i, yellow arrow). Double in situ hybridization studies shows that Heyl (blue) and Wt1 (brown, podocytes) do not overlap (k), whereas Heyl (blue) overlaps with Pdgfrb (brown) in glomerular mesangium (l). Heyl is also expressed in non-vascular locations, in early nephrons (g,h). Panels j, k and l are from different tissue sections from that of panel h. (m-o)Hopx shows very strong and specific expression in the glomerular and extraglomerular mesangium (o, red arrows), whereas Nkx3-1 expression (p-r) is restricted to smooth muscles of renal arteries and arterioles (r, red arrow). (Ba-g) Sfpi1 and Egr1 displayed punctate expression throughout the kidneys, the former reflecting interstitial macrophages and the latter potentially reflecting dynamic signaling responses. Scale bar: 200mm. comprises inner layers of condensed mesenchyme from which the where plasma filtration occurs, the peritubular capillaries at sites of smooth muscle is forming (Yu et al., 2002) and outer layers of tubular reabsorption and secretion, or pericyte-like mesangial cells loose mesenchyme whose fates and functions are poorly and renin-secreting juxtaglomerular cells closely associate with understood. Several genes are documented to show specific blood vessels in or about the renal corpuscle, regulating blood expression in inner condensed mesenchyme (Airik et al., 2006; Nie pressure, glomerular blood flow and glomerular filtration. We et al., 2010; Yu et al., 2002), but no specific markers have been identified a group of 36 transcriptional regulators that displayed a reported for the outer loose mesenchyme. Interestingly, Prrx1, a vascular-related whole-mount in situ hybridization expression paired-related gene, displayed a whole-mount in situ pattern (Fig. 2, supplementary material Table S1). Remarkably, hybridization expression pattern indicative of superficial expression these genes exhibited diverse expression patterns, suggesting in the ureter (Fig. 3E). Section in situ hybridization analysis considerable spatial or temporal heterogeneity within renal confirmed Prrx1 expression was restricted to the outermost layers vasculature-associated cell types. of ureteral mesenchyme, providing a molecular inroad to this cell Bcl6b, which encodes a zinc-finger required for the population (Fig. 3E). enhanced level of the secondary response of memory CD8(+) T Renal vasculature closely associates with nephron components cells (Manders et al., 2005), marked isolated cells in the renal and plays a particularly important role in renal physiology; for cortex and a chain of cells invading the lower cleft of the S-shaped

example, the capillary network within the Bowman’s capsule, body, probably of endothelial nature (Fig. 4Ac). Expression was DEVELOPMENT 1870 RESEARCH ARTICLE Development 139 (10)

Fig. 5. Prediction and validation of transcriptional targets using synexpression and bioinformatics. (A)Genespring histogram representing all genes predicted to show a pattern of synexpression in comparison with the transcription factor Tcfap2b when compared with the 15 developing kidney subcompartments previously profiled by Brunskill et al. (Brunskill et al., 2008). (B)List of the most statistically robust putative targets for each of five transcription factors as assessed using Monkey. (C)An example of validation of such predicted targets. The transcription factor Tcfap2b displays a complex pattern of expression during kidney development, initiating at the ureteric tip (UT) and in the distal renal vesicle (RV) (top panels), distal comma-shaped body, and distal and medial S-shaped body (SSB, middle panels), then later in connecting ducts, distal tubules and the loop of Henle (LoH, lower panels). The same patterns of expression are observed for two predicted targets, Wfdc2 and Pou3f3. CD, collecting duct; CM, cap mesenchyme; CSB, comma-shaped body; CT, connecting tubule; PA, pretubular aggregate; PT, proximal tubule; RC, renal corpuscle; Scale bars: 25mm.

also evident in the glomerular capillaries of the capillary loop stage which encodes a homeobox factor, was expressed in the smooth renal corpuscle (stage III) with expression declining by the muscles surrounding the renal arteries and arterioles (Fig. 4Aq,r). immature renal corpuscle stage (stage IV) (Fig. 4Ab,d). Bcl6b However, in contrast to both Hopx and Heyl, it was not expressed expression was also observed in the endothelium of the peritubular in the mesangium (Fig. 4Ar). capillaries, renal arterioles and renal arteries, but not in renal veins Although the majority of expression patterns could be classified (Fig. 4Ae,f). Expression of Bcl6b in endothelial cells was as including one or more of the eight reference expression patterns, confirmed through colocalization analysis with the endothelial cell we also identified two genes with punctate expression patterns marker Pecam1 (data not shown). throughout the kidney (Fig. 4Ba,d). Sfpi1 (PU.1), a macrophage In the non-endothelial components of the renal vasculature, Heyl lineage marker, displayed a punctate expression pattern was expressed both in smooth muscles surrounding the renal concentrated in the outer cortex, the nascent renal medulla and the arteries and arterioles, and in the glomerular mesangium at the renal corpuscle (Fig. 4Bb,c). This expression pattern agrees with capillary loop stages (stage III) (Fig. 4Ah-l). Its expression in the the previously described interstitial location of resident glomerular mesangium at the immature renal corpuscle stages macrophages visualized using the Csf1r-EGFP transgenic mice (stage IV) is greatly reduced to almost undetectable levels (Fig. (Rae et al., 2007). Egr1 (early growth response 1), a gene involved 4Ai). Co-expression with pericytes/mesangium marker Pdgfrb in cell proliferation, differentiation, stress response (Herschman, confirmed Heyl expression in the glomerular mesangium (Fig. 1991; Liu et al., 1996), stem cell dormancy and stem cell 4Al). Hopx, a homeodomain-containing transcriptional repressor localization (Min et al., 2008), is reported to be upregulated in required for cardiac development that displayed a late tubule specific segments of the kidney epithelia and in the glomerular tuft expression pattern by whole-mount in situ hybridization, was after ischemia-reperfusion (Bonventre et al., 1991). In the E15.5 shown by section in situ hybridization to be expressed in the kidney, Egr1 was expressed sporadically in diverse cell types but mesangium, including both the glomerular mesangium and enriched in the nephrogenic zone, the nascent renal medulla and extraglomerular mesangium, but not in the smooth muscles near or within the (Fig. 4Bf,g). Given that Egr1 is a

surrounding the renal arteries or arterioles (Fig. 4An,o). Nkx3-1, gene that is responsive to diverse growth factor signals, the pattern DEVELOPMENT Transcription factors in kidney RESEARCH ARTICLE 1871 likely reflects active signaling in these regions. The identification of these spotted expression patterns representing single cells scattered throughout the organ demonstrates the resolution and sensitivity of whole-mount in situ hybridization.

Predicting target genes for specific transcriptional regulators defining specific developmental compartments or processes Temporally or spatially restricted transcription factor activity presumably underpins a functional role in distinct developmental processes within disparate cell types in the developing kidney. Having identified novel spatially restricted patterns of transcription factor expression, we sought to identify potential transcription factor-target relationships. Five transcription factors displaying distinct patterns of spatial expression were selected for the analyses: Pou3f1 (late tubule), Tcfap2b (early tubule, and distal and medial segments of the S-shaped body), Sox8 (ureteric tip), Foxi1 (intercalated cells of collecting duct) and Irx1 (early tubule and the medial segment of the S-shaped body). Initially, the expression of each transcription factor was compared across kidney development with previously published expression profiling of the developing kidney generated as another component of the GUDMAP initiative (Brunskill et al., 2008). Only genes displaying microarray-based synexpression matching expression of the canonical transcription factors across all 15 subcompartments of the developing kidney were selected as potential transcription factor targets (Fig. 5A, supplementary material Table S2). For each set of potential target genes, minimal promoters were defined and evidence sought for enriched transcription factor binding within these minimal promoters by the Monkey algorithm (Moses et al., 2004). Monkey analysis assesses Fig. 6. Foxi1 is negatively correlated with its predicted target evolutionary conservation of transcription factor-binding sites, and gene Gsdmc. (A)Section in situ hybridization analysis of four predicted provides a statistical assessment of motif enrichment over chance. Foxi1 targets, Cldn8, Ehf, Gsdmc and Rnf186, showing expression in a Monkey analysis was performed comparing mouse with two region of the cells within the ureteric trunk epithelium. p, pelvis. Scale distinct orthologous groups: rat, guinea pig and rabbit, and rat, bar: 50mm. (B)Two-color single molecule fluorescent in situ human and zebrafish. Fig. 5B lists all predicted targets (with a P- hybridization showing the negative correlation of expression patterns value <10–5) for each transcription factor examined in both between Foxi1 and Gsdmc. Fixed E15.5 kidney sections were ortholog groups. Table S3 in the supplementary material lists the simultaneously hybridized with two differentially labeled probe libraries (Cy5 and Alexa594). Single mRNA molecules appear as diffraction- full analysis including the number of predicted binding sites in each limited spots in an epifluorescence microscope. Transcripts were analysis and the statistical significance of top site predictions. Table automatically detected and assigned to individual cells based on S4 in the supplementary material lists the sequence and statistical membrane staining via myristoylated GFP. Images show detected dots significance of those predicted target sites in the putative target for Foxi1/Gsdmc in seven z-sections at 0.3mm intervals and a promoters for both ortholog comparisons. Section in situ correlation plot for Foxi1/Gsdmc. Each dot in the correlation plot hybridization analysis of the complex synexpression of two denotes the absolute transcript density of a single cell for both genes. predicted target genes of Tcfap2b, Wfdc2 and Pou3f3, with Tcfap2b (C)Model of transcriptional role for Foxi1 in the switch between multi- (Fig. 5C) provides evidence in support of this approach. The subtle potential progenitor and principal cell versus intercalated cell (IC) fate. distinctions in LOH and S-shaped body expression between Tcfap2b and Pou3f3 would argue that other transcription factors are also involved in Pou3f3 regulation. to be regulated by Foxi1 in tissues outside the kidney (ear, Identification of a potential inhibitory role for cartilage, skeleton), section in situ hybridization expression of each Foxi1 in the suppression of the principal cell putative target was compared with data for whole embryo phenotype expression at E14.5 available through Eurexpress Of the chosen regulatory factors for synexpression-target (www.eurexpress.org) (Diez-Roux et al., 2011). Four targets prediction, the best understood is Foxi1, a determinant of showed synexpression with Foxi1 beyond kidney development intercalated cells within the collecting duct epithelium (Blomqvist (Clnd8, Ehf, Dsc2 and Pde8). Section in situ hybridization was et al., 2004). Intercalated cells comprise a distinct, dispersed performed on a subset of putative targets (Cldn8, Ehf, Gsdmc, differentiated cell type that is crucial for maintenance of acid-base Rnf128). Importantly, all showed renal expression restricted to a homeostasis. A total of 52 potential targets were bioinformatically subset of cells in the collecting duct epithelium (Fig. 6A; data not identified through synexpression; Monkey identified putative shown), but in a larger subset of cells than Foxi1, reminiscent of targets in 14 of these (P-value of <10–5 in both ortholog group the water-salt regulating principal cell component of the collecting

analyses). On the assumption that a genuine target is more likely duct epithelium (Fig. 6A). DEVELOPMENT 1872 RESEARCH ARTICLE Development 139 (10)

Table 1. Summary of single-molecule fluorescence in situ hybridization analysis FISH signal Cell number Percentage of total cells Percentage of positive cells Foxi1 and Gsdmc 66 15 20.1 Foxi1 65 14.8 19.8 Gsdmc 197 44.8 60.1 No signal 112 25.4 N/A Total 440 N/A, not applicable.

To examine this possible inverse relationship further, we used unclear, but it is reasonable to anticipate that investigating these single molecule RNA fluorescence in situ hybridization on E15.5 issues will provide new insights into how regulatory programs in kidney sections to label Foxi1 and one of these putative target developing organs generate distinct physiological outputs in the genes, Gsdmc; this approach allows quantitative measurements of functional organ system. up to three different target RNAs in the same sections with single- cell resolution (Raj et al., 2008). Analysis of Foxi1 and Gsdmc Acknowledgements The authors thank Dave Tang and Han Chiu for their technical assistance, Jane transcripts in prospective medullary ureteric trunk epithelial cells Brennan for updating the bioinformatic information of the transcriptional (number of cells440) (processed data, Fig. 6B; raw data, see regulator list, Dr Ariel Gomez for help with annotation of vascular genes, and supplementary material Fig. S1) indicated a significant negative Alison Lee for the mouse transcription factor list. correlation in their expression domains (Spearman correlation coefficient R–0.59, P<10–4). Of the 328 transcript-positive cells Funding M.H.L. is a Principal Research Fellow of the National Health and Medical expressing either gene, 80% showed a mutually exclusive Research Council. The work was supported by a National Institutes of Health expression pattern, i.e. the vast majority of cells either expressed (NIH)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Foxi1 or Gsdmc alone (Fig. 6B and Table 1). These data support a grant [5U01 DK070181 to A.P.M. and DK070136 to M.H.L.]. T.L.B. and P.M. model in which Foxi1 directly silences Gsdmc to elicit intercalated were supported by an NIH R01 grant [RR021692]. Q.R. and J.Y. were cell fate determination. This is likely to also be the case for the supported by a NIH/NIDDK grant [1R01DK085080]. Deposited in PMC for other Foxi1 targets identified in this way. release after 12 months. Competing interests statement DISCUSSION The authors declare no competing financial interests. We have performed a comprehensive, stage-specific analysis of the expression of mouse transcriptional regulators by in situ Supplementary material Supplementary material available online at hybridization in the developing urogenital system. Our goal – to http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.74005/-/DC1 use this regulatory subset of the mammalian genome to identify new cell markers for known cell types, identify novel cellular References heterogeneity and develop predictive insights into regulatory Airik, R., Bussen, M., Singh, M. K., Petry, M. and Kispert, A. (2006). Tbx18 regulates the development of the ureteral mesenchyme. J. Clin. Invest. 116, interactions at play in the developing kidney – was realized in this 663-674. study. Our analysis focused on a single stage of development Bertram, J. F., Douglas-Denton, R. N., Diouf, B., Hughson, M. D. and Hoy, W. identifying a subgroup of specifically expressed transcriptional E. (2011). Human nephron number: implications for health and disease. Pediatr. regulators. Extending the analysis of this group to later stages and Nephrol. 26, 1529-1533. Blomqvist, S. R., Vidarsson, H., Fitzgerald, S., Johansson, B. R., Ollerstam, intersection of this data with other large-scale expression atlases A., Brown, R., Persson, A. E., Bergstrom, G. G. and Enerback, S. (2004). [e.g. Eurexpress (www.eurexpress.org) and the Allen Brain Atlas Distal renal tubular acidosis in mice that lack the forkhead transcription factor (www.brain-map.org)] is likely to provide further information on Foxi1. J. Clin. Invest. 113, 1560-1570. Bonventre, J. V., Sukhatme, V. P., Bamberger, M., Ouellette, A. J. and Brown, cell diversity within the kidney and target relationships in other D. (1991). Localization of the protein product of the immediate early growth organ systems. Importantly, whereas we have focused our response gene, Egr-1, in the kidney after ischemia and reperfusion. Cell Regul. characterization on the kidney, all structures within the urogenital 2, 251-260. dataset have been annotated and these supply a wealth of new Brunskill, E. W., Aronow, B. J., Georgas, K., Rumballe, B., Valerius, M. T., Aronow, J., Kaimal, V., Jegga, A. G., Yu, J., Grimmond, S. et al. (2008). information for secondary analysis of gonads, reproductive ducts Atlas of gene expression in the developing kidney at microanatomic resolution. and lower urinary tract anatomy. The annotated resource is publicly Dev. Cell 15, 781-791. available incorporated within the GUDMAP initiative database Carroll, T. J., Park, J. S., Hayashi, S., Majumdar, A. and McMahon, A. P. (www.gudmap.org). Together, these data will provide valuable (2005). Wnt9b plays a central role in the regulation of mesenchymal to epithelial transitions underlying organogenesis of the mammalian urogenital system. Dev. information and a hypothesis-generating resource for the Cell 9, 283-292. biomedical community. Cebrian, C., Borodo, K., Charles, N. and Herzlinger, D. A. (2004). Our screen identified a large set of transcriptional regulatory genes Morphometric index of the developing murine kidney. Dev. Dyn. 231, 601-608. Costantini, F. and Kopan, R. (2010). Patterning a complex organ: branching that are expressed in spatially or temporally restricted patterns in the morphogenesis and nephron segmentation in kidney development. Dev. Cell 18, developing kidney. The examination of the function of these genes, 698-712. the development of genetic tools enabled by these genes and the Diez-Roux, G., Banfi, S., Sultan, M., Geffers, L., Anand, S., Rozado, D., analysis of synexpression groups overlapping transcription factor- Magen, A., Canidio, E., Pagani, M., Peluso, I. et al. (2011). A high-resolution anatomical atlas of the transcriptome in the mouse embryo. PLoS Biol. 9, defined cellular compartments will provide further insights into the e1000582. molecular networks and cell-cell interactions underpinning kidney Dressler, G. R. (2009). Advances in early kidney specification, development and organogenesis. Initial analysis indicates novel tissue boundaries and patterning. Development 136, 3863-3874. cell groups marked by the expression of transcriptional regulators. Georgas, K., Rumballe, B., Wilkinson, L., Chiu, H. S., Lesieur, E., Gilbert, T. and Little, M. H. (2008). Use of dual section mRNA in situ hybridisation/

The relationship of these domains to structures in the adult kidney is immunohistochemistry to clarify gene expression patterns during the early DEVELOPMENT Transcription factors in kidney RESEARCH ARTICLE 1873

stages of nephron development in the embryo and in the mature nephron of Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. and Luo, L. (2007). A global the adult mouse kidney. Histochem. Cell Biol. 130, 927-942. double-fluorescent Cre reporter mouse. Genesis 45, 593-605. Gomez, R. A., Norwood, V. F. and Tufro-McReddie, A. (1997). Development of Nakai, S., Sugitani, Y., Sato, H., Ito, S., Miura, Y., Ogawa, M., Nishi, M., the kidney vasculature. Microsc. Res. Tech. 39, 254-260. Jishage, K., Minowa, O. and Noda, T. (2003). Crucial roles of Brn1 in distal Gray, P. A., Fu, H., Luo, P., Zhao, Q., Yu, J., Ferrari, A., Tenzen, T., Yuk, D. I., tubule formation and function in mouse kidney. Development 130, 4751-4759. Tsung, E. F., Cai, Z. et al. (2004). Mouse brain organization revealed through Newburger, D. E. and Bulyk, M. L. (2009). UniPROBE: an online database of direct genome-scale TF expression analysis. Science 306, 2255-2257. protein binding microarray data on protein-DNA interactions. Nucleic Acids Res. Grobstein, C. (1953). Inductive epithelio-mesenchymal interaction in cultured 37, D77-D82. organ rudiments of the mouse. Science 118, 52-55. Nie, X., Sun, J., Gordon, R. E., Cai, C. L. and Xu, P. X. (2010). SIX1 acts Grobstein, C. (1955). Inductive interaction in the development of the mouse synergistically with TBX18 in mediating ureteral smooth muscle formation. metanephros. J. Exp. Zool. 130, 319-339. Development 137, 755-765. Hawkins, J., Grant, C., Noble, W. S. and Bailey, T. L. (2009). Assessing Park, J. S., Valerius, M. T. and McMahon, A. P. (2007). Wnt/beta-catenin phylogenetic motif models for predicting transcription factor binding sites. signaling regulates nephron induction during mouse kidney development. Bioinformatics 25, i339-i347. Development 134, 2533-2539. Herschman, H. R. (1991). Primary response genes induced by growth factors and Piper, M., Barry, G., Hawkins, J., Mason, S., Lindwall, C., Little, E., Sarkar, A., tumor promoters. Annu. Rev. Biochem. 60, 281-319. Smith, A. G., Moldrich, R. X., Boyle, G. M. et al. (2010). NFIA controls Kobayashi, A., Valerius, M. T., Mugford, J. W., Carroll, T. J., Self, M., Oliver, telencephalic progenitor cell differentiation through repression of the Notch G. and McMahon, A. P. (2008). Six2 defines and regulates a multipotent self- effector Hes1. J. Neurosci. 30, 9127-9139. renewing nephron progenitor population throughout mammalian kidney Portales-Casamar, E., Thongjuea, S., Kwon, A. T., Arenillas, D., Zhao, X., development. Cell Stem Cell 3, 169-181. Valen, E., Yusuf, D., Lenhard, B., Wasserman, W. W. and Sandelin, A. Koeppen, B. M. and Stanton, B. A. (2001). Renal Physiology. St Louis, MO: (2010). JASPAR 2010: the greatly expanded open-access database of Mosby. transcription factor binding profiles. Nucleic Acids Res. 38, D105-D110. Lee, A. P., Yang, Y., Brenner, S. and Venkatesh, B. (2007). TFCONES: a database Rae, F., Woods, K., Sasmono, T., Campanale, N., Taylor, D., Ovchinnikov, D. of vertebrate transcription factor-encoding genes and their associated conserved A., Grimmond, S. M., Hume, D. A., Ricardo, S. D. and Little, M. H. (2007). noncoding elements. BMC Genomics 8, 441. Characterisation and trophic functions of murine embryonic macrophages based Little, M. H., Brennan, J., Georgas, K., Davies, J. A., Davidson, D. R., Baldock, upon the use of a Csf1r-EGFP transgene reporter. Dev. Biol. 308, 232-246. R. A., Beverdam, A., Bertram, J. F., Capel, B., Chiu, H. S. et al. (2007). A Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A. and Tyagi, S. high-resolution anatomical ontology of the developing murine genitourinary tract. Gene Expr. Patterns 7, 680-699. (2008). Imaging individual mRNA molecules using multiple singly labeled probes. Liu, C., Calogero, A., Ragona, G., Adamson, E. and Mercola, D. (1996). EGR-1, Nat. Methods 5, 877-879. the reluctant suppression factor: EGR-1 is known to function in the regulation of Rumballe, B., Georgas, K. and Little, M. H. (2008). High-throughput paraffin growth, differentiation, and also has significant tumor suppressor activity and a section in situ hybridization and dual immunohistochemistry on mouse tissues. mechanism involving the induction of TGF-beta1 is postulated to account for Cold Spring Harb. Protoc. 5030, 1. this suppressor activity. Crit. Rev. Oncog. 7, 101-125. Saxen, L. (1987). Organogenesis of the Kidney. New York: Cambridge University Manders, P. M., Hunter, P. J., Telaranta, A. I., Carr, J. M., Marshall, J. L., Press. Carrasco, M., Murakami, Y., Palmowski, M. J., Cerundolo, V., Kaech, S. M. Song, R. and Yosypiv, I. V. (2011). Genetics of congenital anomalies of the et al. (2005). BCL6b mediates the enhanced magnitude of the secondary kidney and urinary tract. Pediatr. Nephrol. 26, 353-364. response of memory CD8+ T lymphocytes. Proc. Natl. Acad. Sci. USA 102, Thiagarajan, R. D., Georgas, K. M., Rumballe, B. A., Lesieur, E., Chiu, H. S., 7418-7425. Taylor, D., Tang, D. T., Grimmond, S. M. and Little, M. H. (2011). Matys, V., Kel-Margoulis, O. V., Fricke, E., Liebich, I., Land, S., Barre-Dirrie, Identification of anchor genes during kidney development defines ontological A., Reuter, I., Chekmenev, D., Krull, M., Hornischer, K. et al. (2006). relationships, molecular subcompartments and regulatory pathways. PLoS ONE TRANSFAC and its module TRANSCompel: transcriptional gene regulation in 6, e17286. eukaryotes. Nucleic Acids Res. 34, D108-D110. Woolf, A. S. and Loughna, S. (1998). Origin of glomerular capillaries: is the Min, I. M., Pietramaggiori, G., Kim, F. S., Passegue, E., Stevenson, K. E. and verdict in? Exp. Nephrol. 6, 17-21. Wagers, A. J. (2008). The transcription factor EGR1 controls both the Yu, J., Carroll, T. J. and McMahon, A. P. (2002). Sonic hedgehog regulates proliferation and localization of hematopoietic stem cells. Cell Stem Cell 2, 380- proliferation and differentiation of mesenchymal cells in the mouse metanephric 391. kidney. Development 129, 5301-5312. Moses, A. M., Chiang, D. Y., Pollard, D. A., Iyer, V. N. and Eisen, M. B. (2004). Yu, J., Carroll, T. J., Rajagopal, J., Kobayashi, A., Ren, Q. and McMahon, A. P. MONKEY: identifying conserved transcription-factor binding sites in multiple (2009). A Wnt7b-dependent pathway regulates the orientation of epithelial cell alignments using a binding site-specific evolutionary model. Genome Biol. 5, division and establishes the cortico-medullary axis of the mammalian kidney. R98. Development 136, 161-171. DEVELOPMENT